THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 276, No. 35, Issue of August 31, pp. 32395–32398, 2001
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in U.S.A.
Minireview
How Membranes Shape Protein
Structure*
Published, JBC Papers in Press, June 29, 2001,
DOI 10.1074/jbc.R100008200
Stephen H. White‡, Alexey S. Ladokhin§,
Sajith Jayasinghe, and Kalina Hristova
From the Department of Physiology and Biophysics and
the Program in Macromolecular Structure, University of
California, Irvine, California 92697-4560
Constitutive ␣-helical membrane proteins (MPs)1 are assembled
in membranes by means of a translocation/insertion process that
involves the translocon complex (1). After release into the membrane’s bilayer fabric, a MP resides stably in a thermodynamic free
energy minimum (evidence reviewed in Refs. 2 and 3). This means
that the prediction of MP structure from the amino acid sequence is
fundamentally a problem of physical chemistry, albeit a complex one.
Physical influences that shape MP structure include interactions of
the polypeptide chains with water, each other, the bilayer hydrocarbon core, the bilayer interfaces, and cofactors (Fig. 1). Two recent
reviews (3, 4) provide extensive discussions of the evolution, structure, and thermodynamic stability of MPs. Here we provide a distilled (and updated) overview that addresses four broad questions.
What is the nature of the bilayer matrix that encloses MPs? How can
the thermodynamic principles of MP stability be discovered? How
does the bilayer matrix induce structure? How can the structure of
MPs be predicted? We focus primarily on ␣-helical proteins, but the
thermodynamic principles we present also apply to ␤-barrel MPs,
which Lukas Tamm discusses elsewhere in this series.
Two influences will emerge as paramount in shaping MP structure. First, as implied in Fig. 1, the bilayer fabric of the membrane
has two chemically distinct regions: hydrocarbon core (HC) and
interfaces (IFs). Interfacial structure and chemistry must be important, because the specificity of protein signaling and targeting
by membrane-binding domains could not otherwise exist (5). Second, the high energetic cost of dehydrating the peptide bond, as
when transferring it to a non-polar phase, causes it to dominate in
the formation of structure (6). The only permissible transmembrane structural motifs of MPs are ␣-helices and ␤-barrels, because
internal H-bonding ameliorates this cost.
The Shaping Bilayer Milieu
Because membranes must be in a fluid state for normal cell
function, only the structure of fluid (L␣-phase) bilayers is relevant
to understanding how membranes mold proteins. However, atomic
resolution images of fluid membranes are precluded because of
their high thermal disorder. Nevertheless, useful structural information can be obtained from multilamellar bilayers (liquid crystals) dispersed in water or deposited on surfaces. Their one-dimensional crystallinity allows the distribution of matter along the
bilayer normal to be determined by combined x-ray and neutron
diffraction measurements (liquid crystallography; reviewed in
Refs. 7 and 8)). The resulting “structure” consists of a collection of
* This minireview will be reprinted in the 2001 Minireview Compendium,
which will be available in December, 2001. This is the first article of four in
the “Membrane Protein Structural Biology Minireview Series.” This work
was supported in part by National Institutes of Health Grant GM46823.
‡ To whom correspondence should be addressed: Dept. of Physiology and
Biophysics, University of California, Med. Sci. I D346, Irvine, CA 926974560. Tel.: 949-824-7122; Fax: 949-824-8540; E-mail: [email protected].
uci.edu.
§ Permanent address: Inst. of Molecular Biology and Genetics, National
Academy of Sciences of Ukraine, Kiev 252143, Ukraine.
1
The abbreviations used are: MP, membrane protein; TM, transmembrane; IF, interface; HC, hydrocarbon core; DOPC, dioleoylphosphatidylcholine; POPC, palmitoyloleoylphosphatidylcholine.
This paper is available on line at http://www.jbc.org
time-averaged probability distribution functions of water and lipid
component groups (carbonyls, phosphates, etc.), representing projections of three-dimensional motions onto the bilayer normal (9,
10). The liquid crystallographic structure of an L␣-phase dioleoylphosphatidylcholine (DOPC) bilayer is shown in Fig. 2A (11).
Three features of this structure are important. First, the widths
of the probability densities reveal the great thermal disorder of
fluid membranes. Second, the combined thermal thicknesses of the
IFs (defined by the distribution of the waters of hydration) is about
equal to the 30-Å thickness of the HC. The thermal thickness of a
single IF (⬃15 Å) can easily accommodate an ␣-helix parallel to the
membrane plane (Fig. 2B). The common cartoons of bilayers that
assign a diminutive thickness to the bilayer IFs are thus misleading. Third, the thermally disordered IFs are highly heterogeneous
chemically. As the regions of first contact, the IFs are especially
important in the folding and insertion of non-constitutive MPs,
such as toxins (12), and to the activity of surface-binding enzymes,
such as phospholipase A2 (13). But they are also important in
shaping MP structure (Fig. 1).
A molecule moving from water to the bilayer HC must experience a dramatic variation in environmental polarity over a short
distance because of interfacial chemical heterogeneity, as illustrated by the yellow curve of Fig. 2B (14). An amphipathic helix
such as melittin (15), represented schematically in Fig. 2B, locates
(16) at the midpoint of the steep descent of the polarity gradient.
Because the polarity changes over a distance corresponding roughly
to helix diameter, peptide-bilayer interaction energies must be very
sensitive to polarized helices, such as amphipathic ones.
Coming to Thermodynamic Terms with Insoluble
Membrane Proteins
Experimental exploration of the stability of intact MPs is problematic because of their general insolubility. One approach to stability is to “divide and conquer” by studying the membrane interactions of fragments of MPs, i.e. peptides. Because MPs are
equilibrium structures, folding and stability can be examined by
constructing thermodynamic pathways (3) such as those shown in
Fig. 3. Although these pathways do not mirror the actual biological
assembly process of MPs, they are nevertheless useful for guiding
biological experiments, because they provide a thermodynamic context within which biological processes must proceed.
The four-step model (3) of Fig. 3 is a logical combination of an
early three-step model of Jacobs and White (17) and the two-stage
model of Popot and Engelman (18, 19) in which TM helices are first
“established” across the membrane and then assemble into functional structures (helix association). The model summarizes the
types of experiments on MP folding now being pursued in several
laboratories.
In Fig. 3, the free energy reference state is taken as the unfolded
protein in an IF. However, this state cannot actually be achieved
with MPs because of the solubility problems, nor can it be achieved
with small non-constitutive membrane-active peptides, such as
melittin, because binding usually induces secondary structure
(partitioning-folding coupling). Thus, as is often the case in solution
thermodynamics, the reference state must be a virtual one. It can
be defined for phosphocholine IFs by means of an experimental
interfacial free energy (hydrophobicity) scale (20) derived from the
partitioning into POPC bilayers of tri- and pentapeptides (17, 20)
that have no secondary structure in the aqueous or interfacial
phases. This scale, which includes the peptide bonds as well as the
side chains, allows calculation of the virtual free energy of transfer
of an unfolded chain into an IF. For peptides that cannot form
regular secondary structure, such as the antimicrobial peptide
indolicidin (21), the scale predicts observed free energies of transfer
with remarkable accuracy (22). This validates it for the computation of virtual free energies for partitioning into phosphocholine
IFs. Similar scales are needed for other lipids and lipid mixtures.
32395
32396
Minireview: How Membranes Shape Protein Structure
FIG. 1. Polypeptide interactions that determine the structure and
stability of MPs. The blue lines represent schematically the total thickness
of the lipid bilayer, and the red lines the central hydrocarbon core bounded
by the interfacial regions (see Fig. 2). The protein and lipids shown are from
the 1.55-Å crystallographic structure of bacteriorhodopsin obtained by
Leucke et al. (68) (PDB code 1C3W). Besides interactions of the polypeptide
chain with itself, water, neighboring lipids, and the membrane interface, the
thermodynamic and electrostatic properties of the lipid bilayer itself are
important (3). Interactions with cofactors, such as retinal in the case of
bacteriorhodopsin (purple atoms in the figure), are also important. The lipid
bilayer, like proteins, resides in a free energy minimum resulting from
numerous interactions. This equilibrium can be disturbed by the introduction of proteins or other solutes, resulting in so-called bilayer effects (69),
which also include solvent properties peculiar to bilayers that arise from
motional anisotropy and chemical heterogeneity.
How Membranes Induce Structure: The Importance of the
Peptide Bond
The high cost of interfacial partitioning of the peptide bond (20),
1.2 kcal mol⫺1, explains the origin of partitioning-folding coupling
and also why the interface is a potent catalysis of secondary structure formation. Wimley et al. (23) showed for interfacial ␤-sheet
formation that H-bond formation reduces the cost of peptide partitioning by about 0.5 kcal mol⫺1 per peptide bond. The folding of
melittin into an amphipathic ␣-helix on POPC membranes involves
a per residue reduction of about 0.4 kcal mol⫺1 (24). The folding of
the antimicrobial peptide magainin on charged bilayers seems to
entail a smaller per residue value, about 0.1 kcal mol⫺1 (25). The
cumulative effect of these relatively small per residue free energy
reductions can be very large when tens or hundreds of residues are
involved, as in the assembly of the ␤-barrel transmembrane domain
(26) of ␣-hemolysin that buries ⬃100 residues in the membrane.
Determination of the energetics of TM ␣-helix insertion, which is
critically important for predicting structure, is difficult because
non-polar helices tend to aggregate in both the aqueous and interfacial phases (27). Several efforts have been made, with mixed
success (27–31). Although precise values for the free energy of helix
insertion remain to be established, the broad energetic issues are
clear (32). Computational studies (33, 34) suggest that the transfer
free energy ⌬GCONH of a non-H-bonded peptide bond from water to
alkane is ⫹6.4 kcal mol⫺1, compared with only ⫹2.1 kcal mol⫺1 for
the transfer free energy ⌬GHbond of an H-bonded peptide bond. The
per residue free energy cost of disrupting H-bonds in a membrane
is therefore about 4 kcal mol⫺1. A 20-amino acid TM helix would
cost 80 kcal mol⫺1 to unfold within a membrane, which explains
why unfolded polypeptide chains cannot exist in a transmembrane
configuration.
Fig. 4 illustrates the importance of ⌬GHbond in setting the threshold for transmembrane stability as well as the so-called decision
level in hydropathy plots (35). Using the single membrane-spanning helix of glycophorin A (36) as an example, panel A shows that
the free energy of transfer of the side chains dramatically favors
helix insertion, whereas the transfer cost of the helical backbone
dramatically disfavors insertion. Panel B shows that an uncertainty of 0.5 kcal mol⫺1 in the per residue cost of backbone insertion
has a major effect on the interpretation of hydropathy plots and on
the establishment of the minimum value of side chain hydrophobicity required for transmembrane helix stability. What is the most
likely estimate of ⌬GHbond? The practical number, in the context of
helix
transferring a single glycyl unit of a
Fig. 4A, is the cost of ⌬Gglycyl
polyglycine ␣-helix into the bilayer HC. Electrostatic calculations
(34) and the octanol partitioning study of Wimley et al. (37) suggest
helix
⫽ ⫹1.25 kcal mol⫺1, which is the basis for ⌬Gbb in Fig.
that ⌬Gglycyl
FIG. 2. Liquid crystallographic structure of a fluid lipid bilayer
and its computed polarity profile. The figure is adapted from White and
Wimley (3, 14, 70). A, structure of a DOPC bilayer (5.4 waters/lipid) determined by joint refinement of x-ray and neutron diffraction data (11); B,
polarity profile (yellow curve) of the DOPC bilayer computed from the absolute values of atomic partial charges (14). The end-on view in panel B of an
␣-helix with a diameter of ⬃10 Å (typical for MP helices (46)) shows the
approximate location of the helical axes of the amphipathic helix peptides
Ac-18A-NH2 (71) and melittin (16), as determined by a novel, absolute scale
x-ray diffraction method (reviewed in Ref. 72). The “structure” of the bilayer
shown in panel A is comprised of a collection of transbilayer Gaussian
probability distribution functions representing the lipid components that
account for the entire contents of the bilayer unit cell. The areas under the
curves correspond to the number of constituent groups per lipid represented
by the distributions (1 phosphate, 2 carbonyls, 2 methyls, etc.). The widths of
the Gaussians measure the thermal motions of the lipid components and are
simply related to crystallographic B-factors (9, 16, 71). The thermal motion
of the bilayer is extreme: lipid component B-factors are typically ⬃150 Å2,
compared with ⬃30 Å2 for atoms in protein crystals. In addition to this
thermal motion, two other features of the bilayer are important for shaping
membrane protein structure. First, the IFs have a combined thickness equal
to that of the hydrocarbon core (⬃30 Å). A 15-Å-thick IF can comfortably
accommodate an MP helix lying parallel to the membrane plane. Second, the
IFs are chemically heterogeneous. Panel A shows that they are composed of
water, choline, phosphate, glycerol, carbonyls, and even some methylenes
that spill into the IFs because of thermal motion. Panel B reveals steep
gradients of polarity in the IFs that change over a distance approximately
equal to the diameter of an ␣-helix.
4A. Interestingly, the cost of transferring a random-coil glycyl unit
into n-octanol (37) is ⫹1.15 kcal mol⫺1. This suggests that the
n-octanol whole-residue hydrophobicity scale (3) derived from the
partitioning data of Wimley et al. (37) may be a good measure of
helix
⌬Gglycyl
and therefore useful for identifying ␣-helical TM segments
in hydropathy plots of MPs (3). This is borne out by work in
progress2 using the recently developed MPtopo data base of MPs of
known topology (38), accessible via the World Wide Web
(blanco.biomol.uci.edu/mptopo).
The hydrophobic effect is generally considered to be the major
driving force for compacting soluble proteins (39). However, it
cannot be the force driving compaction (association) of TM ␣-helices. Because the hydrophobic effect arises solely from dehydration
of a non-polar surface (40), it is expended after helices are established across the membrane. Helix association is most likely driven
primarily by van der Waals forces, more specifically the London
dispersion force (reviewed in Refs. 3 and 4), but why would van der
Waals forces be stronger between helices than between helices and
lipids?
Extensive work (41– 45) on dimer formation of glycophorin A in
detergents reveals the answer: knob-into-hole packing that allows
2
Jayasinghe, S., Hristova, K., and White, S. H., (2001) J. Mol. Biol., in
press.
Minireview: How Membranes Shape Protein Structure
FIG. 3. Schematic representation of the shaping of protein structure through polypeptide-bilayer interactions. The figure is based
upon the four-step thermodynamic cycle of White and Wimley (3) for describing the partitioning, folding, insertion, and association of ␣-helical polypeptides. The aqueous insolubility of membrane proteins, folded or unfolded,
precludes direct determinations of interaction free energies. The only possibility for understanding the energetics of MP stability is through studies of
small, water-soluble peptides (20, 23, 24, 27). This approach, summarized in
the figure, uses the unfolded peptide in the IF as the thermodynamic reference state. The free energy of unfolded partitioning in phosphocholine IFs
can now be estimated using the whole-residue interfacial hydrophobicity
scale of Wimley and White (20). Unfolded peptides are driven toward the
folded state in the IF because hydrogen bond formation dramatically lowers
the cost of peptide bond partitioning, which is the dominant determinant of
whole-residue partitioning. The free energy reduction accompanying secondary structure formation is typically ⬃0.4 kcal mol⫺1 per residue (23, 24) but
may be as low as 0.1 kcal mol⫺1 (74). Although small, such changes in
aggregate can be large. For example, the folding of 12 residues of 26-residue
melittin into an ␣-helical conformation causes the folded state to be favored
over the unfolded state by ⬃5 kcal mol⫺1. To put this number in perspective,
the ratio of folded to unfolded peptide is ⬃4700. The cost of partitioning the
peptide bond also dominates transmembrane helix insertion (Fig. 4). The
association of TM helices is probably driven by van der Waals interactions,
giving rise to knob-into-hole packing (43– 45, 75). The GXXXG motif is
especially important in helix-helix interactions in membranes (49, 50).
more efficient packing between helices than between helices and
lipids. Tight, knob-into-hole packing has been found to be a general
characteristic of helical bundle MPs as well (46, 47). For glycophorin A dimerization, knob-into-hole packing is facilitated by the
GXXXG motif, in which the glycines permit close approach of the
helices. The substitution of larger residues for glycine prevents the
close approach and hence dimerization (41, 44, 45). The so-called
TOX-CAT method (48) has made it possible to sample the amino
acid motifs preferred in helix-helix association in membranes by
using randomized sequence libraries (49). The GXXXG motif is
among a significant number of motifs that permit close packing. A
statistical survey of MP sequences disclosed that these motifs are
very common in membrane proteins (50).
Dimerization studies of glycophorin in detergent micelles (44) do
not permit the absolute free energy of association to be determined
because of the large free energy changes associated with micelle
stability. However, estimates (3) suggest 1–5 kcal mol⫺1 as the free
energy cost of separating a helix from a helix bundle within the
bilayer environment. The cost of breaking H-bonds within the
bilayer HC (above) implies that H-bonding between ␣-helices could
provide a strong stabilizing force for helix association. This is borne
out by recent studies of synthetic TM peptides designed to hydrogen bond to one another (51, 52). Interhelical H-bonds, however,
are not common in MPs (reviewed in Ref. 3). Indeed, lacking the
specificity of knobs-into-hole packing, they could be hazardous
because of their tendency to cause promiscuous aggregation (4).
However, they are probably important in the association of transmembrane signaling proteins (53).
32397
FIG. 4. The energetics of transmembrane helix insertion and the
consequences for hydropathy plot analysis. Panel A is based upon
Wimley and White (27). A, estimated relative free energy contributions of the
side chains (⌬Gsc) and backbone (⌬Gbb) to the helix insertion energetics of
glycophorin A (36); B, hydropathy plots of the L subunit of the photosynthetic
reaction center of Rhodobacter sphaeroides showing the importance of knowing the correct value of ⌬Gbb (the green horizontal lines identify the known
transmembrane helices (76)). In panel A, the net side chain contribution
(relative to glycine) was computed using the n-octanol hydrophobicity scale
of Wimley et al. (37). The per residue cost (⌬⌬Gbb) of partitioning a polyglycine ␣-helix can be estimated from the theoretical work of Honig and colleagues (34, 77) to be ⫹1.25 kcal mol⫺1. The cost of partitioning an unfolded
glycyl unit into n-octanol is ⫹1.15 kcal mol⫺1, suggesting that the wholeresidue n-octanol scale (37) provides a reasonable estimate of the free energy
of inserting ␣-helical amino acid residues into bilayers. An exact value of
⌬⌬Gbb is essential for placing hydropathy plots on an absolute thermodynamic scale, which is necessary for distinguishing TM from non-TM peaks in
hydropathy plots. This is shown in panel B. The blue, black, and red curves
are plots made using ⌬⌬Gbb ⫽ 0.75, 1.25, and 1.75 kcal mol⫺1 per residue,
respectively. If ⌬⌬Gbb is too small, TM helices will be overpredicted; if too
large, they will be underpredicted.
Predicting the Structure of Helical Membrane Proteins
As for soluble proteins, the ultimate solution to the problem of
predicting three-dimensional structure of MPs from sequence will
come from a deep quantitative understanding of the energetics of
protein folding. The experimental approaches described above lead
in that direction. At a simple level, the prediction of MP topology is
fairly easy and reliable because of the high hydrophobicity of TM
helices. Such sequence segments are generally apparent in hydropathy analysis (Fig. 4B), which is now a standard prediction tool
(reviewed in Ref. 35). However, the reliability of the resulting
topologies depends strongly upon the hydrophobicity scale used,
and there are many (mostly side chain only scales). An analysis2
using the MPtopo data base (38) reveals that side chain only scales
significantly overpredict TM segments because of the neglect of
⌬Gbb for reasons illustrated by Fig. 4B. The experiment-based
whole-residue hydrophobicity scale of White and Wimley (3), which
takes ⌬Gbb into account, greatly reduces overprediction.2 Membrane Protein Explorer (MPEx) is a Web-based hydropathy analysis tool using this scale (blanco.biomol.uci.edu/mpex). The incorpo-
32398
Minireview: How Membranes Shape Protein Structure
ration into prediction algorithms of additional knowledge of MP
structure and stability, such as the so-called positive-inside rule
(54, 55) or secondary structure propensity (56), can improve the
reliability of topology prediction algorithms. Statistical algorithms
that rely in part on alignment of MP sequences with significant
homology to a sequence of interest can also improve accuracy
(57– 61).
Perspectives
Considerable progress has been made during the past 15 years in
understanding the physical principles underlying MP structure
and stability. Of great importance is the growing number of MPs
whose structures have been determined to high resolution (an
up-to-date list is maintained at blanco.biomol.uci.edu/Membrane_Proteins_xtal.html). About 40 structures have now been
published, and all are either helical bundles or ␤-barrels. An important question is whether new motifs will emerge. Whatever they
may be, they would have to include H-bonded peptide bonds in the
transmembrane segments. One possibility is the ␤-helix motif (62).
A significant feature of many big MPs, such as sarcoplasmic reticulum calcium ATPase (63), is large extracellular domains. This
means that the prediction of MP structure will depend as well upon
success in predicting the structure of soluble proteins. Another
feature not included in any prediction algorithm is the arrangement of subunits, which are common in large MPs.
More information about the assembly of MPs by the translocon
apparatus may result in new insights into structure determination.
New insights are also likely to result from our growing understanding of the role of lipids in MP folding (reviewed in Refs. 64 and 65).
Finally, a more detailed understanding of specific molecular interactions, particularly in mixed-lipid bilayers, will clarify how membrane interfaces shape protein structure. Of particular importance
are the interactions of aromatic residues (66) and charged residues
(67), and how hydrophobic and electrostatic interactions combine to
stabilize proteins at interfaces (22).
Acknowledgments—We thank Michael Myers for editorial assistance. We
are especially pleased to recognize the influential contributions of Dr. William Wimley to the work of our laboratory and to many of the ideas expressed
in this review.
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